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Abstract Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype.

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.

The aim of this study was to investigate coenzyme Q10 (CoQ 10 ) biosynthesis pathway defects in inherited retinal dystrophy. Individuals affected by inherited retinal dystrophy (IRD) underwent exome or genome sequencing for molecular diagnosis of their condition. Following negative IRD gene panel analysis, patients carrying biallelic variants in CoQ 10 biosynthesis pathway genes were identified. Clinical data were collected from the medical records. Haplotypes harbouring the same missense variant were characterised from family genome sequencing (GS) data and direct Sanger sequencing. Candidate splice variants were characterised using Oxford Nanopore Technologies single molecule sequencing. The CoQ 10 status of the human plasma was determined in some of the study patients. 13 individuals from 12 unrelated families harboured candidate pathogenic genotypes in the genes: PDSS1, COQ2, COQ4 and COQ5. The PDSS1 variant c.589 A > G was identified in three affected individuals from three unrelated families on a possible ancestral haplotype. Three variants (PDSS1 c.468-25 A > G, PDSS1 c.722-2 A > G, COQ5 c.682-7 T > G) were shown to lead to cryptic splicing. 6 affected individuals were diagnosed with non-syndromic retinitis pigmentosa and 7 had additional clinical findings. This study provides evidence of CoQ 10 biosynthesis pathway gene defects leading to non-syndromic retinitis pigmentosa in some cases. Intronic variants outside of the canonical splice-sites represent an important cause of disease. RT-PCR nanopore sequencing is effective in characterising these splice defects., (© 2022. The Author(s).)

Background: The molecular genetic basis of pulmonary arterial hypertension (PAH) is heterogeneous, with at least 26 genes displaying putative evidence for disease causality. Heterozygous variants in the ATP13A3 gene were recently identified as a new cause of adult-onset PAH. However, the contribution of ATP13A3 risk alleles to child-onset PAH remains largely unexplored., Methods and Results: We report three families with a novel, autosomal recessive form of childhood-onset PAH due to biallelic ATP13A3 variants. Disease onset ranged from birth to 2.5 years and was characterised by high mortality. Using genome sequencing of parent-offspring trios, we identified a homozygous missense variant in one case, which was subsequently confirmed to cosegregate with disease in an affected sibling. Independently, compound heterozygous variants in ATP13A3 were identified in two affected siblings and in an unrelated third family. The variants included three loss of function variants (two frameshift, one nonsense) and two highly conserved missense substitutions located in the catalytic phosphorylation domain. The children were largely refractory to treatment and four died in early childhood. All parents were heterozygous for the variants and asymptomatic., Conclusion: Our findings support biallelic predicted deleterious ATP13A3 variants in autosomal recessive, childhood-onset PAH, indicating likely semidominant dose-dependent inheritance for this gene., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Purpose: To report novel genotypes and expand the phenotype spectrum of SSBP1-disease and explore potential disease mechanism., Methods: Five families with previously unsolved optic atrophy and retinal dystrophy underwent whole genome sequencing as part of the National Institute for Health Research BioResource Rare-Diseases and the UK's 100,000 Genomes Project. In silico analysis and protein modelling was performed on the identified variants. Deep phenotyping including retinal imaging and International Society for Clinical Electrophysiology of Vision standard visual electrophysiology was performed., Results: Seven individuals from five unrelated families with bilateral optic atrophy and/or retinal dystrophy with extraocular signs and symptoms in some are described. In total, 6 SSBP1 variants were identified including the previously unreported variants: c.151A>G, p.(Lys51Glu), c.335G>A p.(Gly112Glu), and c.380G>A, p.(Arg127Gln). One individual was found to carry biallelic variants (c.380G>A p.(Arg127Gln); c.394A>G p.(Ile132Val)) associated with likely autosomal recessive SSBP1-disease. In silico analysis predicted all variants to be pathogenic and Three-dimensional protein modelling suggested possible disease mechanisms via decreased single-stranded DNA binding affinity or impaired higher structure formation., Conclusions: SSBP1 is essential for mitochondrial DNA replication and maintenance, with defects leading to a spectrum of disease that includes optic atrophy and/or retinal dystrophy, occurring with or without extraocular features. This study provides evidence of intrafamilial variability and confirms the existence of an autosomal recessive inheritance in SSBP1-disease consequent upon a previously unreported genotype.

Purpose: The purpose of this study was to report retinal dystrophy as a novel clinical feature and expand the ocular phenotype in patients harboring biallelic candidate FDXR variants., Methods: Patients carrying biallelic candidate FDXR variants were identified by whole genome sequencing (WGS) as part of the National Institute for Health Research BioResource rare-disease and the UK's 100,000 Genomes Project (100KGP) with an additional case identified by exome sequencing. Retrospective clinical data were collected from the medical records. Haplotype reconstruction was performed in families harboring the same missense variant., Results: Ten individuals from 8 unrelated families with biallelic candidate variants in FDXR were identified. In addition to bilateral optic atrophy and variable extra-ocular findings, 7 of 10 individuals manifested retinal dystrophy comprising dysfunction and degeneration of both rod and cone photoreceptors. Five of 10 subjects had sensorineural hearing loss. The previously unreported missense variant (c.1115C > A, p.(Pro372His)) was found in 5 of 8 (62.5%) study families. Haplotype reconstruction using WGS data demonstrated a likely ancestral haplotype., Conclusions: FDXR-associated disease is a phenotypically heterogeneous disorder with retinal dystrophy being a major clinical feature observed in this cohort. In addition, we hypothesize that a number of factors are likely to drive the pathogenesis of optic atrophy, retinal degeneration, and perhaps the associated systemic manifestations.

Purpose: Previous studies suggest that ceramide is a proapoptotic lipid as high levels of ceramides can lead to apoptosis of neuronal cells, including photoreceptors. However, no pathogenic variant in ceramide synthases has been identified in human patients and knockout of various ceramide synthases in mice has not led to photoreceptor degeneration., Methods: Exome sequencing was used to identify candidate disease genes in patients with vision loss as confirmed by standard evaluation methods, including electroretinography (ERG) and optical coherence tomography. The vision loss phenotype in mice was evaluated by ERG and histological analyses., Results: Here we have identified four patients with cone-rod dystrophy or maculopathy from three families carrying pathogenic variants in TLCD3B. Consistent with the phenotype observed in patients, the Tlcd3b KO/KO mice exhibited a significant reduction of the cone photoreceptor light responses, thinning of the outer nuclear layer, and loss of cone photoreceptors across the retina., Conclusion: Our results provide a link between loss-of-function variants in a ceramide synthase gene and human retinal dystrophy. Establishment of the Tlcd3b knockout murine model, an in vivo photoreceptor cell degeneration model due to loss of a ceramide synthase, will provide a unique opportunity in probing the role of ceramide in survival and function of photoreceptor cells.

Beyond the major gene BMPR2 , several new genes predisposing to PAH have been identified during the last decade. Recently, preliminary evidence of the involvement of the KDR gene was found in a large genetic association study.We prospectively analysed the KDR gene by targeted panel sequencing in a series of 311 PAH patients referred to a clinical molecular laboratory for genetic diagnosis of PAH.Two index cases with severe PAH from two different families were found to carry a loss-of-function mutation in the KDR gene. These two index cases were clinically characterised by low diffusing capacity for carbon monoxide adjusted for haemoglobin ( D LCO c) and interstitial lung disease. In one family, segregation analysis revealed that variant carriers are either presenting with PAH associated with low D LCO c, or have only decreased D LCO c, whereas non-carrier relatives have normal D LCO c. In the second family, a single affected carrier was alive. His carrier mother was unaffected with normal D LCO c.We provided genetic evidence for considering KDR as a newly identified PAH-causing gene by describing the segregation of KDR mutations with PAH in two families. In our study, KDR mutations are associated with a particular form of PAH characterised by low D LCO c and radiological evidence of parenchymal lung disease including interstitial lung disease and emphysema., Competing Interests: Conflict of interest: M. Eyries has nothing to disclose. D. Montani reports grants and personal fees from Actelion and Bayer, personal fees from GSK, Pfizer and MSD, outside the submitted work. Conflict of interest: B. Girerd has nothing to disclose. Conflict of interest: N. Favrolt has nothing to disclose. Conflict of interest: M. Riou has nothing to disclose. Conflict of interest: L. Faivre has nothing to disclose. Conflict of interest: G. Manaud has nothing to disclose. Conflict of interest: F. Perros has nothing to disclose. Conflict of interest: S. Gräf has nothing to disclose. Conflict of interest: M.W. Morrell is an employee of Morphogen-IX. Conflict of interest: M. Humbert reports personal fees from Acceleron, Actelion, Merck and United Therapeutics, grants and personal fees from Bayer and GSK, outside the submitted work. Conflict of interest: F. Soubrier has nothing to disclose., (Copyright ©ERS 2020.)

Pulmonary arterial hypertension (PAH) is a rare disease that leads to premature death from right heart failure. It is strongly associated with elevated red cell distribution width (RDW), a correlate of several iron status biomarkers. High RDW values can signal early-stage iron deficiency or iron deficiency anaemia. This study investigated whether elevated RDW is causally associated with PAH.A two-sample Mendelian randomisation (MR) approach was applied to investigate whether genetic predisposition to higher levels of RDW increases the odds of developing PAH. Primary and secondary MR analyses were performed using all available genome-wide significant RDW variants (n=179) and five genome-wide significant RDW variants that act via systemic iron status, respectively.We confirmed the observed association between RDW and PAH (OR 1.90, 95% CI 1.80-2.01) in a multicentre case-control study (cases n=642, disease controls n=15 889). The primary MR analysis was adequately powered to detect a causal effect (odds ratio) between 1.25 and 1.52 or greater based on estimates reported in the RDW genome-wide association study or from our own data. There was no evidence for a causal association between RDW and PAH in either the primary (OR causal 1.07, 95% CI 0.92-1.24) or the secondary (OR causal 1.09, 95% CI 0.77-1.54) MR analysis.The results suggest that at least some of the observed association of RDW with PAH is secondary to disease progression. Results of iron therapeutic trials in PAH should be interpreted with caution, as any improvements observed may not be mechanistically linked to the development of PAH., Competing Interests: Conflict of interest: A. Ulrich has nothing to disclose. Conflict of interest: J. Wharton has nothing to disclose. Conflict of interest: T.E. Thayer has nothing to disclose. Conflict of interest: E.M. Swietlik has nothing to disclose. Conflict of interest: T.R. Assad has nothing to disclose. Conflict of interest: A.A. Desai has nothing to disclose. Conflict of interest: S. Gräf has nothing to disclose. Conflict of interest: L. Harbaum has nothing to disclose. Conflict of interest: M. Humbert reports grants and personal fees from Bayer and GSK, personal fees from Actelion, Merck and from United Therapeutics, outside the submitted work. Conflict of interest: N.W. Morrell reports personal fees from Actelion and Morphogen-IX, outside the submitted work. Conflict of interest: W.C. Nichols has nothing to disclose. Conflict of interest: F. Soubrier has nothing to disclose. Conflict of interest: L. Southgate has nothing to disclose. Conflict of interest: D-A. Trégouët has nothing to disclose. Conflict of interest: R.C. Trembath reports personal fees for advisory board work from Ipsen Pharmaceuticals, personal fees for non-executive board membership from King's College Hospital NHS Foundation Trust, outside the submitted work. Conflict of interest: E.L. Brittain reports personal fees for advisory board work from Bayer, outside the submitted work. Conflict of interest: M.R. Wilkins reports grants from Vifor Pharma, outside the submitted work. Conflict of interest: I. Prokopenko has nothing to disclose. Conflict of interest: C.J. Rhodes reports personal fees from Actelion, outside the submitted work., (Copyright ©ERS 2020.)

While traffic and air pollution exposure is associated with increased mortality in numerous diseases, its association with disease severity and outcomes in pulmonary arterial hypertension (PAH) remains unknown.Exposure to particulate matter with a 50% cut-off aerodynamic diameter ≤2.5 μm (PM 2.5 ), nitrogen dioxide (NO 2 ) and indirect measures of traffic-related air pollution (distance to main road and length of roads within buffer zones surrounding residential addresses) were estimated for 301 patients with idiopathic/heritable PAH recruited in the UK National Cohort Study of Idiopathic and Heritable PAH. Associations with transplant-free survival and pulmonary haemodynamic severity at baseline were assessed, adjusting for confounding variables defined a priori Higher estimated exposure to PM 2.5 was associated with higher risk of death or lung transplant (unadjusted hazard ratio (HR) 2.68 (95% CI 1.11-6.47) per 3 μg·m -3 ; p=0.028). This association remained similar when adjusted for potential confounding variables (HR 4.38 (95% CI 1.44-13.36) per 3 μg·m -3 ; p=0.009). No associations were found between NO 2 exposure or other traffic pollution indicators and transplant-free survival. Conversely, indirect measures of exposure to traffic-related air pollution within the 500-1000 m buffer zones correlated with the European Society of Cardiology/European Respiratory Society risk categories as well as pulmonary haemodynamics at baseline. This association was strongest for pulmonary vascular resistance.In idiopathic/heritable PAH, indirect measures of exposure to traffic-related air pollution were associated with disease severity at baseline, whereas higher PM 2.5 exposure may independently predict shorter transplant-free survival., Competing Interests: Conflict of interest: E. Sofianopoulou has nothing to disclose. Conflict of interest: S. Kaptoge reports grants from UK Medical Research Council and British Heart Foundation, during the conduct of the study. Conflict of interest: S. Gräf has nothing to disclose. Conflict of interest: C. Hadinnapola has nothing to disclose. Conflict of interest: C.M. Treacy has nothing to disclose. Conflict of interest: C. Church has nothing to disclose. Conflict of interest: G. Coghlan has nothing to disclose. Conflict of interest: J.S.R. Gibbs reports grants and personal fees from Actelion, Bayer, GSK and MSD, personal fees from Arena, Bellerophon, Complexa and Pfizer, grants from Amco and United Therapeutics, outside the submitted work. Conflict of interest: M. Haimel has nothing to disclose. Conflict of interest: L.S. Howard has nothing to disclose. Conflict of interest: M. Johnson reports grants and personal fees for attendance at meeting and lectures from Actelion, Bayer, GSK and MSD, outside the submitted work. Conflict of interest: D.G. Kiely reports grants, personal fees and nonfinancial support from Actelion, Bayer and GSK, personal fees and nonfinancial support from MSD, outside the submitted work. Conflict of interest: A. Lawrie reports grants and personal fees from Actelion and GSK, grants from British Heart Foundation and UK Medical Research Council, outside the submitted work. Conflict of interest: J. Lordan has nothing to disclose. Conflict of interest: R.V. MacKenzie Ross has nothing to disclose. Conflict of interest: J.M. Martin has nothing to disclose. Conflict of interest: S. Moledina has nothing to disclose. Conflict of interest: M. Newnham reports education support (travel, registration and accommodation) to attend conferences from MSD and GSK, outside the submitted work. Conflict of interest: A.J. Peacock reports grants and personal fees from Actelion and Bayer, personal fees from GSK, grants from Gilead, outside the submitted work. Conflict of interest: L.C. Price has nothing to disclose. Conflict of interest: C.J. Rhodes has nothing to disclose. Conflict of interest: J. Suntharalingam has nothing to disclose. Conflict of interest: E.M. Swietlik has nothing to disclose. Conflict of interest: M.R. Toshner has nothing to disclose. Conflict of interest: J. Wharton reports personal fees for advisory board work from Actelion, outside the submitted work. Conflict of interest: M.R. Wilkins has nothing to disclose. Conflict of interest: S.J. Wort reports grants and personal fees from Actelion and Bayer, personal fees from GSK and MSD, outside the submitted work. Conflict of interest: J. Pepke-Zaba (or her institution) received research, education grants from Actelion, Merck and Bayer; and has served on advisory boards for Actelion, Merck, Bayer and GSK. Conflict of interest: R. Condliffe reports personal fees for advisory board work from Actelion, Bayer and MSD, during the conduct of the study. Conflict of interest: P.A. Corris reports grants and personal fees from Actelion and Bayer, personal fees from MSD, outside the submitted work. Conflict of interest: E. Di Angelantonio reports grants from European Commission Framework 7, European Research Council, British Heart Foundation, UK Medical Research Council and National Institute for Health Research, during the conduct of the study; grants from NHS Blood and Transplant, outside the submitted work. Conflict of interest: S. Provencher has nothing to disclose. Conflict of interest: N.W. Morrell reports personal fees from GSK and Johnson & Johnson/Actelion, outside the submitted work., (Copyright ©ERS 2019.)

Cell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity; however, existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin-associated factors that modulate levels of transcriptional noise. Herein, we show destabilization of pluripotency-associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl-transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5), have been inhibited. Functionally, network destabilization associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we show evidence of a relationship between transcriptional heterogeneity and cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells, suggesting a general principle that could be exploited in other normal and malignant stem cell fate transitions. Stem Cells 2018;36:1828-11., (©2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2018.)

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α 1 -subunit of the voltage-gated Ca V 2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed Ca V 2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: We describe the clinical features in two pedigrees with dominantly inherited retinopathy segregating the previously reported frameshifting mutation, c.836dupG (p.Ile280Asn*78) in the terminal exon of the RGR gene, and compare their haplotypes to that of the previously reported pedigree., Methods: The probands were ascertained at West Virginia University Eye Institute (WVU) and Moorfields Eye Hospital (MEH) through next generation sequencing (NGS) and whole genome sequencing (WGS) respectively. Clinical data included visual acuity (VA), visual fields, fundus autofluorescence (FAF), optical coherence tomography (OCT), and electroretinography (ERG). Haplotype analysis was performed using Sanger sequencing of the DNA from the molecularly ascertained individuals from the three pedigrees., Results: Nine heterozygous mutation carriers were identified in two families. Four carriers were asymptomatic; five carriers had variable VA reduction, visual field constriction, and experienced difficulty under dim illumination. Fundus examination of the asymptomatic carriers showed diffuse or reticular pigmentation of the retina; the symptomatic carriers had chorioretinal atrophy. FAF imaging showed widespread signal loss in advanced retinopathy, and reticular hyperautofluorescence in mild cases. OCT showed loss of outer retinal lamina in advanced disease. ERG showed moderate-to-severe rod-cone dysfunction in two symptomatic carriers; and was normal in three asymptomatic carriers. A shared haplotype flanking the mutation of up to 6.67 Mb was identified in both families. Within this region, 1.27 Mb were shared with the first family reported with this retinopathy., Conclusions: The clinical data suggest a variable and slow degeneration of the RPE. A shared chromosomal segment surrounding the RGR gene suggests a single ancestral mutational event underlying all three families.

Purpose: Mutations in ARL2BP, encoding ADP-ribosylation factor-like 2 binding protein, have recently been implicated as a cause of autosomal recessive retinitis pigmentosa (arRP), with three homozygous variants identified to date. In this study, we performed next-generation sequencing to reveal additional arRP cases associated with ARL2BP variants., Methods: Whole-genome sequencing (WGS) or whole-exome sequencing (WES) was performed in 1,051 unrelated individuals recruited for the UK Inherited Retinal Disease Consortium and NIHR-BioResource Rare Diseases research studies. Sanger sequencing was used to validate the next-generation sequencing data, and reverse transcriptase (RT)-PCR analysis was performed on RNA extracted from blood from affected individuals to test for altered splicing of ARL2BP . Detailed phenotyping was performed, including clinical evaluation, electroretinography, fundus photography, fundus autofluorescence imaging, and spectral-domain optical coherence tomography., Results: Homozygous variants in ARL2BP (NM_012106.3) were identified in two unrelated individuals with RP. The variants, c.207+1G>A and c.390+5G>A, at conserved splice donor sites for intron 3 and intron 5, respectively, were predicted to alter the pre-mRNA splicing of ARL2BP . RT-PCR spanning the affected introns revealed that both variants caused abnormal splicing of ARL2BP in samples from affected individuals., Conclusions: This study identified two homozygous variants in ARL2BP as a rare cause of arRP. Further studies are required to define the underlying disease mechanism causing retinal degeneration as a result of mutations in ARL2BP and any phenotype-genotype correlation associated with residual levels of the wild-type transcript.

To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype-phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull's-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes-intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498_506del, p.(Ile167_Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498_506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date.

Pulmonary arterial hypertension (PAH) is a rare disorder with a poor prognosis. Deleterious variation within components of the transforming growth factor-β pathway, particularly the bone morphogenetic protein type 2 receptor (BMPR2), underlies most heritable forms of PAH. To identify the missing heritability we perform whole-genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses reveal significant overrepresentation of rare variants in ATP13A3, AQP1 and SOX17, and provide independent validation of a critical role for GDF2 in PAH. We demonstrate familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, lead to reduced secretion from transfected cells. In addition, we identify pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these findings contribute new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention.

Background: Diagnostic use of gene panel next-generation sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD., Methods: Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory., Results: We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques., Conclusion: Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations., Competing Interests: Competing interests: CT is an employee of Congenica Ltd. All other authors declare no competing interests., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 ( BMPR2 ) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene ( EIF2AK4 ) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH., Methods: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured., Results: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival., Conclusions: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation., (© 2017 The Authors.)

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

High-profile genomic variation projects like the 1000 Genomes project or the Exome Aggregation Consortium, are generating a wealth of human genomic variation knowledge which can be used as an essential reference for identifying disease-causing genotypes. However, accessing these data, contrasting the various studies and integrating those data in downstream analyses remains cumbersome. The Human Genome Variation Archive (HGVA) tackles these challenges and facilitates access to genomic data for key reference projects in a clean, fast and integrated fashion. HGVA provides an efficient and intuitive web-interface for easy data mining, a comprehensive RESTful API and client libraries in Python, Java and JavaScript for fast programmatic access to its knowledge base. HGVA calculates population frequencies for these projects and enriches their data with variant annotation provided by CellBase, a rich and fast annotation solution. HGVA serves as a proof-of-concept of the genome analysis developments being carried out by the University of Cambridge together with UK's 100 000 genomes project and the National Institute for Health Research BioResource Rare-Diseases, in particular, deploying open-source for Computational Biology (OpenCB) software platform for storing and analyzing massive genomic datasets., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To describe better the motor phenotype, molecular genetic features, and clinical course of GNAO1 -related disease., Methods: We reviewed clinical information, video recordings, and neuroimaging of a newly identified cohort of 7 patients with de novo missense and splice site GNAO1 mutations, detected by next-generation sequencing techniques., Results: Patients first presented in early childhood (median age of presentation 10 months, range 0-48 months), with a wide range of clinical symptoms ranging from severe motor and cognitive impairment with marked choreoathetosis, self-injurious behavior, and epileptic encephalopathy to a milder phenotype, featuring moderate developmental delay associated with complex stereotypies, mainly facial dyskinesia and mild epilepsy. Hyperkinetic movements were often exacerbated by specific triggers, such as voluntary movement, intercurrent illnesses, emotion, and high ambient temperature, leading to hospital admissions. Most patients were resistant to drug intervention, although tetrabenazine was effective in partially controlling dyskinesia for 2/7 patients. Emergency deep brain stimulation (DBS) was life saving in 1 patient, resulting in immediate clinical benefit with complete cessation of violent hyperkinetic movements. Five patients had well-controlled epilepsy and 1 had drug-resistant seizures. Structural brain abnormalities, including mild cerebral atrophy and corpus callosum dysgenesis, were evident in 5 patients. One patient had a diffuse astrocytoma (WHO grade II), surgically removed at age 16., Conclusions: Our findings support the causative role of GNAO1 mutations in an expanded spectrum of early-onset epilepsy and movement disorders, frequently exacerbated by specific triggers and at times associated with self-injurious behavior. Tetrabenazine and DBS were the most useful treatments for dyskinesia.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs ∗ 63), c.1996C>T (p.Arg666 ∗ ), c.2632G>T (p.Glu878 ∗ ), and c.2738&#95;2761del (p.Arg913&#95;Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)